Effects of Temperature and Osmolytes on Competing Degradation Routes for an Ig G1 Antibody.

Addition of excipients is a common strategy to slow protein aggregation during storage. Excipient effects on the mechanism(s) and temperature ( T) dependence of aggregation for a monoclonal antibody solution were tested using size-exclusion chromatography, differential scanning calorimetry ( DSC), temperature scanning monomer loss ( TSML), and laser light scattering; previous work in buffer-only conditions had shown non- Arrhenius behavior and implicated Fab and/or C H3 unfolding as a key step in aggregation. Excipients included citrate, amino acid salts (histidine- HCl, arginine- HCl), and polyols (mannitol and glycerol). DSC and TSML showed that Fab, rather than CH3, unfolding corresponded with the onset of aggregation for each condition. Isothermal incubation at 56.5° C, 40° C, and 2° C-8° C resulted in aggregation, while fragmentation occurred readily at only 40° C. The primary effect of the different excipients appeared to be preferential accumulation/exclusion, affecting the concentrations of partially unfolded monomer key intermediates. In addition, aggregation rates were clearly non- Arrhenius, causing aggregation to dominate over fragmentation at high and low T, and making long-term stability predictions problematic based on commonly employed 40° C conditions. Possible reasons for non- Arrhenius behavior include a strong T-dependence of the Fab unfolding enthalpy and/or a switch from Fab-mediated to Fc-mediated aggregation as one moves from high to low T. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3556-3566, 2013 [ABSTRACT FROM AUTHOR]